Inhibition of glutaminase C

ABSTRACT

The present invention relates to a method of reducing the production of glutamate from glutamine by glutaminase C in a cell or tissue. The method involves inhibiting glutaminase C activity in the cell or tissue under conditions effective to reduce production of glutamate from glutamine. Compounds for carrying out this method are also disclosed and include those of formula (III): 
                         
wherein B, R 1c , R 2c , m, and n are defined herein.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/163,304, filed Mar. 25, 2009, which is herebyincorporated by reference in its entirety.

This invention was made with government support under grant numbers RO1GM40654, RO1 GM47458, and RO1 GM61762 awarded by National Institutes ofHealth. The U.S. Government has certain rights in this invention.

FIELD OF INVENTION

The present invention relates to the inhibition of glutaminase C (GAC).

BACKGROUND OF THE INVENTION

Tumor cells have an absolute requirement for glutamine as a growthsubstrate. Glutamine is required as a precursor for both DNA synthesisand protein synthesis, and may also be used as a respiratory substrate.In experiments where glutamine metabolism in tumor cells has beenspecifically compared with that in non-transformed cells of the sameorigin, glutamine metabolism in the tumor cells has been found to beconsiderably faster. This is true for human hepatocytes and hepatomacells (Souba, W., “Gutamine and Cancer,” Ann. Surg. 218:715-728 (1993))and also for glutamine oxidation in rat kidney fibroblasts and ratfibrosarcoma cells (Fischer et al., “Adaptive Alterations in CellularMetabolism and Malignant Transformation,” Ann. Surg. 227:627-634(1998)).

The first reaction in glutamine metabolism is hydrolysis of glutamine toglutamate via the mitochondrial enzyme phosphate-dependent glutaminase.Two major isoforms of this enzyme have been characterized. These areknown as the kidney form (K-type) which was first cloned from rat kidney(Shapiro et al., “Isolation, Characterisation, and In vitro Expressionof a cDNA That Encodes the Kidney Isoenzyme of the MitochondrialGlutaminase,” J. Biol. Chem. 266:18792-18796 (1991)) and is expressed inmany mammalian tissues, and the liver form (L-type) (Chung-Bok et al.,“Rat Hepatic Glutaminase, Identification of the Full Coding Sequence andCharacterisation of a Functional Promoter,” Biochem. J. 324:193-200(1997)) which was originally identified in post-natal liver. These twoenzymes have different kinetic properties. Although the cDNAs encodingthe two isoforms have regions of high sequence similarity, they alsodiffer significantly elsewhere and the enzyme isoforms are the productsof different genes (for a review see (Curthoys et al., “Regulation ofGlutaminase Activity and Glutamine Metabolism,” Annu. Rev. Nutr.16:133-159 (1995)). Glutamine metabolism is essential for tumor cellgrowth but there are few studies at present on glutaminase expression intumor cells. In mouse Ehrlich ascites cells (Quesada et al.,“Purification of Phosphate-Dependent Glutaminase from IsolatedMitochondria of Ehrlich Ascites-Tumor Cells,” Biochem. J. 255:1031-1035(1988)) and rat fibrosarcoma cells (Fischer et al., “AdaptiveAlterations in Cellular Metabolism and Malignant Transformation,” Ann.Surg. 227:627-634 (1998)), an enzyme with the kinetic properties of theK-type glutaminase is expressed. Rat and human hepatocytes express theL-type glutaminase, but this is not expressed in hepatoma cell lines,which express the K-type instead (Souba, W. W., “Glutamine and Cancer,”Ann. Surg. 218:715-728 (1993)). Inhibition of K-type glutaminaseexpression by anti-sense mRNA in Ehrlich ascites cells has been shown todecrease the growth and tumorigenicity of these cells (Lobo et al.,“Inhibition of Glutaminase Expression by Antisense mRNA Decreases Growthand Tumorigenicity of Tumor Cells,” Biochem. J. 348:257-261 (2000)).

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method of reducingthe production of glutamate from glutamine by glutaminase C in a cell ora tissue. The method involves inhibiting glutaminase C activity in thecell or tissue under conditions effective to reduce production ofglutamate from glutamine.

A second aspect of the present invention relates to a method of treatinga subject with a condition mediated by production of glutamate fromglutamine by glutaminase C. The method involves selecting a subject witha condition mediated by production of glutamate from glutamine andadministering to said selected subject an inhibitor of glutaminase Cactivity under conditions effective to treat the condition mediated byproduction of glutamate from glutamine.

A third aspect of the present invention relates to a pharmaceuticalcomposition comprising a compound selected from the group consisting of:

(i) a compound of formula (II):

wherein:

-   -   n is an integer from 1 to 4;    -   R_(1b) is independently at each occurrence H, OH, OR_(5b),        halogen, CN, NO₂, NH₂, NHR_(5b), NR_(5b)R_(6b), C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen;    -   R_(2b) is independently H, halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl, or mono        or polycyclic aryl;    -   R_(3b) and R_(4b) are independently H, OR_(5b), SR_(5b),        SR_(5b)S(O)—, R_(5b)S(O)₂, —COOR_(5b), —C(O)NR_(5b)R_(6b), C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen; or    -   R_(3b) and R_(4b) can combine together to form a mono or        polycyclic heterocyclyl or heteroaryl containing from 1-5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each formed heteroaryl or heterocyclyl        optionally substituted with substituents selected from the group        consisting of oxo, thio, amino, C₁-C₆ alkyl, C₂-C₆ alkenyl, and        C₂-C₆ alkynyl; and    -   R_(5b) and R_(6b) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each one of R_(5b) or R_(6b) optionally        substituted from 1 to 3 times with substituents selected from        the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, and C₄-C₇ cycloalkylalkyl;

(ii) a compound of formula (III):

wherein:

-   -   m and n are integers from 1 to 4;    -   B is a substituted or unsubstituted mono or polycyclic aryl or        mono or polycyclic heterocyclyl or heteroaryl with each cyclic        unit containing from 1 to 5 heteroatoms selected from the group        consisting of nitrogen, sulfur, and oxygen;    -   R₁, and R_(2c) are independently H, OH, OR_(3c), halogen, CN,        NO₂, COOH, NH₂, NHR_(3c), NR_(3c)R_(4c), C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen; and    -   R_(3c) and R_(4c) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl containing from 1 to 5 heteroatoms selected from the        group consisting of nitrogen, sulfur, and oxygen; and

(iii) a compound comprising the active moiety of formula II or formulaIII.

A fourth aspect of the present invention relates to the compound offormula:

A fifth aspect of the present invention relates to the method ofscreening for compounds capable of reducing the production of glutamatefrom glutamine. The method involves providing a cell or tissue underconditions effective for the cell or tissue to produce glutamate fromglutamine as a result of glutaminase C activity. A plurality ofcandidate compounds is then provided to contact the cell or tissue andthe candidate compounds which inhibit glutaminase C activity as a resultof said contacting are identified.

It has been found that V5-tagged GAC, when ectopically expressed inDbl-transformed cells followed by its immunoprecipitation (IP), exhibitsignificantly higher activity compared to V5-GAC IPed fromnon-transformed NIH 3T3 cells. The GA activity IPed from Dbl-transformedcells is inhibited by the methods as well as the compounds of thepresent invention, and is markedly reduced when NF-kB activation isblocked prior to IP. This is consistent with the suggestion that GAC ismodified in transformed cells in an NF-kB-dependent manner.

Also described is the importance of cellular metabolism in thedevelopment of cancer and, in particular, the early observations thattumor cells exhibit enhanced glycolytic activity (i.e. the “Warburgeffect”). In particular, a novel regulatory connection between the RhoGTPases and the activation of the mitochondrial enzyme glutaminase C isdescribed, thus shedding new light on how glutamine metabolism iselevated in tumorigenesis. These findings raise interestingpossibilities regarding the targeting of the enzyme activity ofglutaminase C as a potential therapeutic strategy against malignanttransformation.

In addition, the present invention offers an entirely novel approach toidentification and development of drugs designed to inhibit the functionof glutaminase C. Since it is well-known that tumorigenesis is linked toglutamine metabolism, the present invention can have an important impactin anti-cancer drug development.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E illustrate that the small molecule 968 inhibiting cellulartransformation. FIG. 1A shows NIH 3T3 cells that are transientlytransfected with oncogenic Dbl and cultured for 14 days, while treatedwith different benzo[a]phenanthridinones (designated 384, 335, 968, 537,and 343) (10 μM each). Cells were fixed and stained with crystal violetfor counting of foci. Right: 968 was serially diluted (10, 5, 2.5, and1.25 μM) and evaluated for its ability to inhibit focus formation. FIG.1B shows NIH 3T3 cells that are stably transfected with Dbl and grown inDMEM supplemented with 1% calf serum and the indicated amounts of 968 orBA-968. After 6 days, the cells are counted. 100% represents the numberof Dbl-transformed cells counted in the absence of 968 (27.5×10⁴ cells).Data represent the average of 3 experiments (±s.d.). FIG. 1C shows thedifferent benzo[a]phenanthridinone derivatives examined for theireffects on Dbl-induced focus formation (designated 968, BA968, 335, 343,031, 537, 5043, and 384). FIG. 1D shows control NIH 3T3 cells that arecultured in DMEM supplemented with 10% calf serum, and either untreatedor treated with 10 μM 968 or 335. At the indicated times, the cells arecounted. Data represent the average of 3 experiments (±s.d.). FIG. 1Eshows photomicrographs of Dbl-transfected NIH 3T3 cells (bottom panels)and control NIH 3T3 cells (top panels) treated with either DMSO or 5 μM968.

FIGS. 2A-G illustrate effects of 968 on the transforming activity ofconstitutively active Rho GTPases and human breast cancer cells. FIG. 2A(top) shows that NIH 3T3 cells stably expressing hemagglutinin(HA)-tagged Cdc42(F28L), Rac(F28L), RhoC(F30L), or vector control cells,either treated with 10 μM 968 or untreated, are grown in soft agar.Cells are scored after 14 days and plotted as the percentage of thetotal number of colonies greater than 50 mm in diameter. Data representthe average of 3 experiments (±s.d.). FIG. 2A (bottom) shows therelative expression of the HA-tagged GTPases. FIG. 2B shows cells thatare treated with 10 μM 968 or untreated, cultured in DMEM supplementedwith 10% calf serum for 6 days, and then counted. Data represent theaverage of 3 experiments (±s.d.). FIG. 2C shows cells that are culturedin DMEM supplemented with 1% calf serum, treated with 10 μM 968 oruntreated, and counted at the indicated times. Data represent theaverage of 3 experiments (±s.d.). FIG. 2D shows cells that areserum-starved, treated with 10 μM 968 or untreated, and seeded inMilliCell upper chambers containing growth factor-reduced Matri-gel.After 24 hours at 37° C., the migratory cells are fixed, stained withGIEMSA, and counted. Data represent the average of 3 experiments(±s.d.). FIG. 2E shows MDA-MB231 cells, SKBR3 cells, and NIH 3T3 cellsstably expressing Dbl that are treated with 10 μM 968 or untreated, andgrown in soft-agar as in FIG. 2A. Data represent the average of 3experiments (±s.d.). FIG. 2F shows breast cancer cells that are culturedin RPMI 1640 medium supplemented with 10% fetal bovine serum, and HMECsare cultured in MEGM complete medium, for 6 days in the presence orabsence of 10 μM 968, and then counted. Data represent the average of 3experiments (±s.d.). FIG. 2G shows breast cancer cells that are culturedin RPMI 1640 medium supplemented with 1% fetal bovine serum, treatedwith 10 μM 968 or untreated, and analyzed as in 2C. Data represent theaverage of 3 experiments (±s.d.).

FIGS. 3A-G show that glutaminase C serves as a target for 968. FIG. 3Ashows the E. coli-expressed mouse ortholog of human GAC that is assayedin the presence or absence of the indicated amounts of 968 (●) or 335 () 100%=620 moles of glutamine hydrolyzed per minute per mole of enzyme.Data represent the average of 3 experiments (±s.d.). FIG. 3A (top panel)shows the biotin-labeled, active moiety of 968 linked tostreptavidin-agarose beads, or control beads alone that is incubatedwith NIH 3T3 cell lysates transiently expressing V5-tagged mouse GAC.Following precipitation of the beads and re-suspension, the samples areanalyzed by Western blotting with anti-V5 antibody. FIG. 3B (left) showsNIH 3T3 cells stably expressing HA-Cdc42(F28L), cells stably expressingHA-Cdc42(F28L) transfected with control siRNA or siRNAs targeting bothisoforms of mouse KGA, or control cells that are grown in DMEMsupplemented with 1% calf serum and counted. Data represent the averageof 3 experiments (±s.d.). FIG. 3B (right) shows the efficiencies ofsiRNAs targeting both isoforms of KGA, and the relative levels ofHA-Cdc42 in the different cells. FIG. 3C (top) shows breast cancer cellsthat are grown in RPMI 1640 medium supplemented with 1% fetal bovinecalf serum. Data represent the average of 3 experiments (±s.d.). FIG. 3C(bottom) shows the relative efficiencies of siRNAs targeting bothisoforms of KGA. FIG. 3D shows SKBR3 cells that are grown in 1% fetalbovine serum as in 3C except in the presence of 10 μM 968 alone ortogether with 7 μM dimethy α-ketoglutarate. Data represent the averageof 3 experiments (±s.d.). FIG. 3E NIH 3T3 cells transiently expressingDbl are assayed for focus formation in the presence of 10 μM 968 aloneor together with 7 μM dimethyl α-ketoglutarate. FIG. 3F (top) showsmitochondrial fractions from equivalent numbers of the indicated stablecell lines, treated or untreated with 10 μM 968 that are assayed forbasal (phosphate-independent) GA activity. 100%=680 nM glutaminehydrolyzed per minute per 10⁵ cells. Data are the average of 3experiments (±s.d.). FIG. 3G (bottom) shows the relative amounts of KGA(using an antibody which recognizes both isoforms) in the mitochondrialpreparations. FIG. 3G (top) shows the mitochondrial fractions fromequivalent numbers of the indicated cells, treated or untreated with 10μM 968, were assayed for basal (phosphate-independent) GA activity.100%=750 nM glutamine hydrolyzed per minute per 10⁵ cells. Datarepresent the average of 3 experiments (±s.d.). FIG. 3G (bottom) showsthe relative amounts of KGA and VDAC present in the mitochondrialpreparations.

FIGS. 4A-E illustrate the role of glutaminase C activity in cellulartransformation. FIG. 4A (left) shows control NIH 3T3 cells, NIH 3T3cells transiently expressing Dbl, cells stably expressing Cdc42(F28L)and transiently expressing mouse GAC, cells stably expressingCdc42(F28L), cells transiently expressing GAC alone, and cells stablyexpressing Cdc42(F28L) and transiently expressing Dbl that are examinedfor focus-forming activity. FIG. 4A (right) shows the quantification offoci. Data represent the average of 3 experiments (±s.d.). FIG. 4B(left) shows the focus-forming assays performed on NIH 3T3 cells stablyexpressing Cdc42(F28L), cells stably expressing Cdc42(F28L) andtransiently expressing Dbl, and cells stably expressing Cdc42(F28L) andeither transiently expressing wild-type mouse GAC or the GAC(S291A)mutant. FIG. 4B (right) shows the quantification of foci. Data representthe average of 3 experiments (±s.d.). FIG. 4C (top) shows the basal(phosphate-independent) GA activity in the mitochondrial fractions fromNIH 3T3 cells stably expressing Dbl that were cultured for 2 days andtreated or untreated with 2 μM BAY 11-7082, or transfected with controlsiRNA or siRNAs targeting the p65/RelA subunit. 100% represents theactivity measured in untreated Dbl-transformed cells. The data representthe average of 2 experiments. FIG. 4C (bottom) shows the relativeamounts of KGA (using an antibody which recognizes both isoforms)present in the mitochondria from the indicated cells, and the relativeefficiencies of two siRNAs targeting p65/RelA. FIG. 4D (top) shows thebasal (phosphate-independent) GA activity in the mitochondrial fractionsfrom HMECs, and SKBR3 cells treated or untreated with 2 μM BAY 11-7082,or transfected with siRNAs targeting p65/RelA. 100% represents theactivity measured in untreated SKBR3 cells. The data represent theaverage of 2 experiments. FIG. 4D (bottom) shows the relative amounts ofKGA (using an antibody which recognizes both isoforms) present in themitochondria from the indicated cells, and the relative efficiencies oftwo siRNAs targeting p65/RelA. FIG. 4E (top) shows that V5-GAC wastransiently expressed in NIH 3T3 cells stably expressing Dbl that weretreated with 2 μM BAY 11-7082 or untreated, or in control NIH 3T3 cells,and then immunoprecipitated and assayed for GA activity in the absenceof phosphate, and in the presence or absence of 10 μM 968 or BA-986.Data represent the average of 3 experiments (±s.d.). FIG. 4E (bottom)shows the relative expression of V5-GAC.

FIGS. 5A-C illustrate comparative abilities of 968 and otherbenzo[a]phenanthridinones to inhibit the transforming activity ofoncogenic Dbl and H-Ras. FIG. 5A shows NIH 313 cells that aretransiently transfected with oncogenic Dbl for 14 days while treatedwith different benzo[a]phenanthridinones (designated 384, 335, 968, 537,343, 031, and 5043, see FIG. 1C for structures) (5 μM, each) that aredissolved in DMSO. Histograms show the relative levels of Dbl-inducedfocus formation for the different treatments, compared to Dbl-inducedfocus formation measured in the presence of DMSO (i.e. solvent control).Data represent the average of 3 experiments (mean±s.d.). FIG. 5B showsNIH 3T3 cells that are transiently transfected with H-Ras(G12V) andcultured for 14 days, while treated with differentbenzo[a}phenanthridinones (5 μM, each) that are dissolved in DMSO.Histograms show the relative levels of Ras(G12V)-induced focus formationmeasured for the different treatments, compared to Ras(G12V)-inducedfocus formation measured in the presence of DMSO (solvent control). Datarepresent the average of 3 experiments (mean±s.d.). FIG. 5C NIH 3T3cells stably expressing H-Ras(G12V) that are cultured in DMEMsupplemented with 1% calf serum, with the indicated amounts of 968.After 6 days, cells are trypsinized and counted. 100% represents thenumber of cells counted in the absence of 968, i.e. 26×10⁴ cells. Datarepresent the average of 3 experiments (mean±s.d.).

FIG. 6 shows that Rho GTPases are hyper-activated in breast cancercells. Lysates from MDA-MB231 cells, SKBR3 cells, and HMECs, areprepared and incubated with GST fused to the limit Rho-binding domain onRhotekin (GST-RBD). The top panels show the relative levels of RhoA-GTPand RhoC-GTP that are co-precipitated with GST-RBD from the indicatedcells, as indicated by Western blotting with an anti-RhoA monoclonalantibody and an anti-RhoC polyclonal antibody. The middle panels comparethe relative expression of RhoA and RhoC in whole cell lysates (WCL)from the different cells and the bottom panel shows the relative inputof GST-RBD.

FIG. 7 illustrates the MS peptide analysis of the silver-stained bandthat was specifically precipitated by the biotin-labeled, active moietyof 968. Shown in the figure is the alignment of mouse kidney-typeglutaminase (KGA) isoform-1 (SEQ ID NO: 1) and isoform-2 (the mouseortholog of human GAC) (SEQ ID NO: 2). The peptides identified by theMaldi-MS analysis of the protein precipitated by the biotin-labeled,active moiety of 968 are in red. The peptide VLSPEAVR (SEQ ID NO: 3) ispresent in both isoforms whereas VSPESSDDTSTTVVYR (SEQ ID NO: 4) maps tothe C-terminus unique to the mouse GAC (Accession #NP_001106854). GAChas a predicted molecular weight of 65,864.

FIG. 8 illustrates that the biotin-labeled, active moiety of 968 bindsto a 66 kDa protein that cross-reacts with the anti-KGA polyclonalantibody. The biotin-labeled, active moiety of 968 linked tostreptavidin-agarose beads, or control beads alone, are incubated withlysates from NIH 3T3 cells stably expressing the constitutively activeCdc42(F28L) mutant. Following precipitation of the beads andre-suspension, the samples are analyzed by SDS-PAGE and silver-staining(left-hand panel), as well as by Western blot analysis using an anti-KGApolyclonal antibody.

FIGS. 9A-C show that 968 is not competitive versus either theGA-substrate, glutamine, nor inorganic phosphate, an allostericactivator, of GA activity. The activity of the E. coli-expressedrecombinant mouse ortholog of human GAC are assayed in the presence of 0(●), 10 (∇) or 20 (▪) μM 968 and inorganic phosphate in the form ofdipotassium hydrogen phosphate. FIG. 9A shows that the concentration ofinorganic phosphate is kept constant at 150 mM and the concentration ofglutamine was varied from 0 to 50 mM. FIG. 9B shows the data in FIG. 9Ais shown as a double-reciprocal lineweaver-burke plot. FIG. 9C showsthat the concentration of glutamine is kept constant at 20 mM and theconcentration of inorganic phosphate has varied from 0 to 200 mM. Thedata are plotted as GA activity as function of varying concentrations ofglutamine or inorganic phosphate. The data are the average of 3experiments and are plotted as mean±SEM.

FIGS. 10A-C illustrate the effects of knocking-down KGA on the growth oftransformed/cancer cells versus NIH 3T3 cells. FIG. 10A shows that NIH313 cells stably expressing Cdc42(F28L), cells stably expressingCdc42(F28L) transfected with control siRNA or siRNAs targeting bothisoforms of mouse KGA, and control (vector) cells, are grown in softagar and scored after 10 days. Histograms show the percentage of thetotal number of colonies greater than 50 μm in diameter. Data representthe average of 3 experiments (mean±s.d.). FIG. 10B shows that NIH 3T3cells are transfected with control siRNA or siRNAs targeting bothisoforms of mouse KGA, cultured in DMEM supplemented with 10% calf serumfor the indicated number of days, and then are trypsinized and counted.FIG. 10B (top) shows histograms that represent the average of 3experiments (mean±s.d.). FIG. 10B (bottom panel) shows the relativeefficiencies of the siRNAs targeting KGA. FIG. 10C shows that theindicated breast cancer cell lines are transfected with control siRNA orwith siRNAs targeting both isoforms of KGA and then grown in soft agarand scored after 10 days as described in S6A. Data represent the averageof 3 experiments (mean±s.d.).

FIGS. 11A-E illustrate the effects of different treatments on GAactivity. FIG. 11A shows that mitochondrial fractions from equivalentnumbers of the indicated stable cell lines, treated or untreated with 10μM 968, are assayed for GA activity in the presence of 133 mM inorganicphosphate (+Pi). The addition of Pi stimulated the GA activity incontrol 3T3 cells, Dbl-expressing cells, and Cdc42(F28L)-expressingcells by ˜6-fold, 2-fold, and 3-fold, respectively. 100% represents thePi-stimulated activity measured for Dbl-transformed cells that are nottreated with 968. Data is the average of 3 experiments (±s.d.). FIG. 11Bshows that mitochondrial fractions from equivalent numbers of theindicated cells, treated or untreated with 10 μM 968, are assayed for GAactivity in the presence of 133 mM Pi. The addition of Pi stimulated theGA activity of HMECs, MDA-MB231 cells, and SKBR3 cells by ˜5-fold,2-fold, and 1.4-fold, respectively. 100% represents the Pi-stimulatedactivity for SKBR3 cells that were not treated with 968. Data are theaverage of 3 experiments (±s.d.). FIG. 11C (left) shows that SKBR3 cellsare transfected with control siRNA or siRNAs targeting the RhoA and RhoCGTPases (i.e. a double knock-down). Mitochondrial fractions are preparedfrom equal numbers of cells and assayed for GA activity in the presenceor absence of 133 mM Pi. The data are plotted as the percentage of GAactivity measured for untreated SKBR3 cells and represent the average of2 experiments. FIG. 11C (right) shows that the efficiencies of thesiRNAs against RhoC and RhoA were assessed by Western blot analysisusing anti-RhoA and anti-RhoC antibodies. FIG. 11D shows Pi-stimulatedGA activity in mitochondrial fractions from NIH 3T3 cells stablyexpressing Dbl that were cultured for 2 days and treated or untreatedwith 2 μM BAY 11-7082, or transfected with control siRNA or siRNAstargeting the p65/RelA subunit. 100% represents the P1-stimulatedactivity measured for untreateDbl-transformed cells. The data representthe average of 2 experiments. FIG. 11E shows Pi-stimulated GA activityin the mitochondrial fractions from SKBR3 cells treated with 2 μM BAY11-7082, or transfected with control siRNA or siRNAs targeting p65/RelA.100% represents the Pi-stimulated activity for untreated SKBR3 cells.The data represent the average of 2 experiments.

FIG. 12 illustrates GAC expression levels in normal and cancerous breasttissues obtained from 80 patients.

FIG. 13 shows that GAC, but not KGA, mRNA levels are increased in highergrade breast tumors.

FIG. 14 shows that GAC, but not KGA, enhances the oncogenic potential ofCdc42.

FIG. 15 illustrates that GAC is differentially phosphorylated intransformed (Dbl) cells but not in normal NIH 3T3 cells.

FIG. 16 illustrates that the phosphorylation of GAC is necessary for itsbasal glutaminase activity.

FIG. 17 illustrates that 968 treatment of cells inhibits the formationof at least one phosphorylation on GAC.

FIG. 18 illustrates a model for the mode of action of 968 on GAC andoncogenic growth.

FIG. 19 illustrates both 968 and BA-968 are effective inhibitors of GACactivity.

DETAILED DESCRIPTION OF THE INVENTION

As used above, and throughout the description of this invention, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings. If not defined otherwise herein, all technicaland scientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. In the event that there is a plurality of definitions for aterm herein, those in this section prevail unless stated otherwise.

The term “halo” or “halogen” means fluoro, chloro, bromo, or iodo.

The term “optionally substituted” indicates that a group may have asubstituent at each substitutable atom of the group (including more thanone substituent on a single atom), and the identity of each substituentis independent of the others.

The term “substituted” or “substitution” of an atom means that one ormore hydrogen on the designated atom is replaced with a selection fromthe indicated group, provided that the designated atom's normal valencyis not exceeded. “Unsubstituted” atoms bear all of the hydrogen atomsdictated by their valency. When a substituent is oxo (i.e., ═O), then 2hydrogens on the atom are replaced. Combinations of substituents and/orvariables are permissible only if such combinations result in stablecompounds; by “stable compound” or “stable structure” is meant acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. Exemplary substitutents include, withoutlimitation, oxo, thio (i.e. ═S), nitro, cyano, halo, OH, NH₂, C₁-C₆alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₄-C₇ cycloalkylalkyl, monocyclic aryl, monocyclic heteroaryl,polycyclic aryl, and polycyclic heteroaryl.

The term “monocyclic” indicates a molecular structure having one ring.

The term “polycyclic” indicates a molecular structure having two or morerings, including, but not limited to, fused, bridged, or spiro rings.

The term “alkyl” means an aliphatic hydrocarbon group which may bestraight or branched having about 1 to about 6 carbon atoms in thechain. Branched means that one or more lower alkyl groups such asmethyl, ethyl or propyl are attached to a linear alkyl chain. Exemplaryalkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl,t-butyl, n-pentyl, and 3-pentyl.

The term “alkenyl” means an aliphatic hydrocarbon group containing acarbon-carbon double bond and which may be straight or branched havingabout 2 to about 6 carbon atoms in the chain. Preferred alkenyl groupshave 2 to about 4 carbon atoms in the chain. Branched means that one ormore lower alkyl groups such as methyl, ethyl, or propyl are attached toa linear alkenyl chain. Exemplary alkenyl groups include ethenyl,propenyl, n-butenyl, and i-butenyl.

The term “alkynyl” means an aliphatic hydrocarbon group containing acarbon-carbon triple bond and which may be straight or branched havingabout 2 to about 6 carbon atoms in the chain. Preferred alkynyl groupshave 2 to about 4 carbon atoms in the chain. Branched means that one ormore lower alkyl groups such as methyl, ethyl, or propyl are attached toa linear alkynyl chain. Exemplary alkynyl groups include ethynyl,propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.

The term “alkoxy” means an alkyl-O—, alkenyl-O—, or alkynyl-O— groupwherein the alkyl, alkenyl, or alkynyl group is described above.Exemplary alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy,n-butoxy, pentoxy, and hexoxy.

The term “cycloalkyl” refers to a non-aromatic saturated or unsaturatedmono- or polycyclic ring system which may contain 3 to 6 carbon atoms;and which may include at least one double bond. Exemplary cycloalkylgroups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.

The term “cycloalkylalkyl” refers to a radical of the formula—R^(a)R^(b) where R^(a) is an alkyl radical as defined above and R^(b)is a cycloalkyl radical as defined above. The alkyl radical and thecycloalkyl radical may be optionally substituted as defined above.

The term “aryl” refers to aromatic monocyclic or polycyclic ring systemcontaining from 6 to 19 carbon atoms, where the ring system may beoptionally substituted. Aryl groups of the present invention include,but are not limited to, groups such as phenyl, naphthyl, azulenyl,phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl,chrysenyl, and naphthacenyl.

The term “arylalkyl” refers to a radical of the formula —R^(a)R^(b)where R^(a) is an alkyl radical as defined above and R^(b) is an arylradical as defined above. The alkyl radical and the cycloalkyl radicalmay be optionally substituted as defined above.

The term “aryarylalkyl” refers to a radical of the formula—R^(a)R^(b)R^(c) where R^(a) is an alkyl as defined above, R^(b) is anaryl radical as defined above, and R^(c) is an aryl radical as definedabove. The alkyl radical and both aryl radicals may be optionallysubstituted as defined above.

The term “heterocyclyl” refers to a stable 3- to 18-membered ringradical which consists of carbon atoms and from one to five heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur. Forpurposes of this invention, the heterocyclyl radical may be amonocyclic, or a polycyclic ring system, which may include fused,bridged, or spiro ring systems; and the nitrogen, carbon, or sulfuratoms in the heterocyclyl radical may be optionally oxidized; thenitrogen atom may be optionally quaternized; and the ring radical may bepartially or fully saturated. Examples of such heterocyclyl radicalsinclude, without limitation, azepinyl, azocanyl, pyranyl dioxanyl,dithianyl, 1,3-dioxolanyl, tetrahydrofuryl, dihydropyrrolidinyl,decahydroisoquinolyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolidinyl,oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl,pyrazolidinyl, thiazolidinyl, tetrahydropyranyl, thiamorpholinyl,thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone.

The term “heteroaryl” refers to an aromatic ring radical which consistsof carbon atoms and from one to five heteroatoms selected from the groupconsisting of nitrogen, oxygen, and sulfur. For purposes of thisinvention the heteroaryl may be a monocyclic or polycyclic ring system;and the nitrogen, carbon, and sulfur atoms in the heteroaryl ring may beoptionally oxidized; the nitrogen may optionally be quaternized.Examples of heteroaryl groups include, without limitation, pyrrolyl,pyrazolyl, imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl,pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl,furopyrrolyl, indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl,indazolyl, benzimidazolyl, imidazopyridinyl, benzotriazolyl,benzoxazolyl, benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl,triazolopyridinyl, thienopyridinyl, benzothiadiazolyl, benzofuyl,benzothiophenyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl,tetrahydroisoquinolyl, cinnolinyl, quinazolinyl, quinolizilinyl,phthalazinyl, benzotriazinyl, chromenyl, naphthyridinyl, acrydinyl,phenanzinyl, phenothiazinyl, phenoxazinyl, pteridinyl, and purinyl.

Further heterocycles and heteraryls are described in Katritzky et al.,eds., “Comprehensive Heterocyclic Chemistry: The Structure, Reactions,Synthesis and Use of Heterocyclic Compounds,” Vol. 1-8, Pergamon Press,N.Y. (1984), which is hereby incorporated by reference in its entirety.

The term “compounds of the present invention”, and equivalentexpressions are meant to embrace compounds of general Formulae (I),(II), and/or (III) (as well as compounds comprising their activemoieties) as herein before described, which expression includes theprodrugs, the pharmaceutically acceptable salts, and the solvates, e.g.,hydrates, where the context so permits. Similarly, reference tointermediates, whether or not they themselves are claimed, is meant toembrace their salts and solvates, where the context so permits. For thesake of clarity, particular instances, when the context so permits, aresometimes indicated in the text, but these instances are purelyillustrative and it is not intended to exclude other instances when thecontext so permits.

The term “treatment” or “treating” means any manner in which one or moreof the symptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use for treating diseases ordisorders mediated by the production of glutamate from glutamine.

This invention also envisions the “quaternization” of any basicnitrogen-containing groups of the compounds disclosed herein. The basicnitrogen can be quaternized with any agents known to those of ordinaryskill in the art including, for example, lower alkyl halides, such asmethyl, ethyl, propyl and butyl chloride, bromides and iodides; dialkylsulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides including benzyl and phenethylbromides. Water or oil-soluble or dispersible products may be obtainedby such quaternization.

A first aspect of the present invention relates to a method of reducingthe production of glutamate from glutamine in a cell or a tissue. Themethod involves inhibiting glutaminase C activity in the cell or tissueunder conditions effective to reduce production of glutamate fromglutamine.

Glutaminase C is the isoform-2 of the human glutaminase, an enzyme foundin kidney and other tissues and generally referred as kidney-typeglutaminase. Glutaminase C is involved in the hydrolysis of glutamine toglutamate and ammonium.

In one embodiment, this aspect of the present invention can be carriedout by inhibiting overexpression-independent glutaminase C activityand/or inhibiting glutaminase C activity independent of exogenousphosphate addition. Alternatively, an activating phosphorylation eventon glutaminase C can be inhibited. As a further alternative of themethod of the present invention, inhibition of glutaminase C activitycan be performed by inhibiting glutaminase C hyperactivity.

Although glutaminase C expression has been found to be increased in somecancers, applicants have found that the participation of GAC is notlimited to an increase in expression. Some cancer cells (such as thebreast cancer cell line, SKBR3) have been found to exhibit GACexpression levels which are similar to normal cells, but are stilldependent on the presence of GAC for cell growth (see FIG. 3C). Thus, byreducing the normal expression levels of GAC, one can inhibit GACactivity in cancer cells.

GAC isolated from cancer cells can show an elevated glutaminase activitylevel relative to GAC isolated from normal cells when assayed in theabsence of phosphate, but in the presence of phosphate the enzymesisolated from both normal and cancer cells show a similar extent ofactivation per amount of GAC (FIG. 3G and FIG. 11B). Thus, the GAC incancer cells is not dependent on the exogenous addition of phosphate tobe active Inhibition of the phosphate-independent activation of GAC incancer cells would inhibit the production of glutamate from glutamine.

One way in which the GAC activity from cancer cells may be increasedrelative to the GAC activity in normal cells is by a phosphorylationevent that occurs on GAC. If the phosphorylations on GAC areremoved/blocked using either alkaline phosphate or a small molecule(e.g., compound 968 in FIG. 1C), the ability for GAC to produceglutamate from glutamine is limited.

The activation state of GAC may vary among different cancer cells,regardless of the expression levels of GAC. A higher amount of activitymay be referred to as “hyperactivity”. For example, Dbl transformedcells and Cdc42 F28L transformed cells contain similar levels of GAC asdo untransformed NIH 3T3 cells. However, the GAC in the Dbl and Cdc42transformed cells shows a higher activation than in the non-transformedcells, with the GAC from the Dbl cells being approximately twice asactive than the GAC from the Cdc42 transformed cells (FIG. 3F). Thus,the GAC in the Dbl transformed cells is hyperactive Inhibiting thehyperactivity of GAC in Dbl cells would limit the production ofglutamate from glutamine by glutaminase C.

In another embodiment of this aspect of the present invention, themethod of inhibiting involves providing a compound selected from thegroup consisting of:

(i) a compound of formula (I):

wherein:

-   -   the dotted circle identifies an active moiety;    -   X is independently —CR_(14a)— or N;    -   R_(1a) is independently H, OH, OR_(14a), C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, R_(14a)C(O)—, R_(14a)OC(O)—,        R_(14a)S(O)—, or R_(14a)S(O)₂—;    -   R_(2a), R_(3a), R_(4a), R_(5a), and R_(6a) are each        independently H, halogen, NO₂, OH, OR_(14a), —SR_(14a), NH₂,        NHR_(14a), NR_(14a)R_(15a), R_(14a)C(O)—, R_(14a)OC(O)—,        R_(14a)C(O)O—, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkenyl, C₃-C₆        cycloalkyl, C₄-C₇ cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or        polycyclic aryl, or mono or polycyclic heteroaryl with each        cyclic unit containing from 1 to 5 heteroatoms selected from the        group consisting of nitrogen, sulfur, and oxygen; or    -   R_(2a) and R_(3a), R_(3a) and R_(4a), R_(4a) and R_(5a), or        R_(5a) and R_(6a) can combine to form a heterocyclic ring;    -   R_(7a), R_(8a), R_(9a), and R_(10a) are each independently H,        OH, NH₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆        cycloalkyl, C₄-C₇ cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or        polycyclic aryl, or mono or polycyclic heteroaryl with each        cyclic unit containing from 1 to 5 heteroatoms selected from the        group consisting of nitrogen, sulfur, and oxygen, wherein the        aryl, heteroaryl, and aryl C₁-C₆ alkyl are optionally        substituted from 1 to 3 times with substitutents selected from        the group consisting of halogen, OH, NH₂, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₁-C₆ alkoxy, SH, and C₁-C₆ thioalkyl; and    -   R_(11a), R_(12a), R_(13a), R_(14a), R_(15a), R_(16a), and        R_(17a) are each independently H, halogen, OH, NO₂, C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, each        one of R_(11a)-R_(17a) optionally substituted with NH₂, OH,        halogen, COOH, NO₂, and CN;

(ii) a compound of formula (II):

wherein:

-   -   the dotted circle identifies an active moiety;    -   n is an integer from 1 to 4;    -   R_(1b) is independently at each occurrence H, OH, OR_(5b),        halogen, CN, NO₂, NH₂, NHR_(5b), NR_(5b)R_(6b), C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen;    -   R_(2b) is independently H, halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl, or mono        or polycyclic aryl;    -   R_(3b) and R_(4b) are independently H, OR_(5b), SR_(5b),        R_(5b)S(O)—, R_(5b)S(O)₂—, —COOR_(5b), —C(O)NR_(5b)R_(6b), C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen; or    -   R_(3b) and R_(4b) can combine together to form a mono or        polycyclic heterocyclyl or heteroaryl containing from 1-5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each formed heteroaryl or heterocyclyl        optionally substituted with substituents selected from the group        consisting of oxo, thio, amino, C₁-C₆ alkyl, C₂-C₆ alkenyl, and        C₂-C₆ alkynyl; and    -   R_(5b) and R_(6b) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each one of R_(5b) or R_(6b) optionally        substituted from 1 to 3 times with substituents selected from        the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, and C₄-C₇ cycloalkylalkyl;

(iii) a compound of formula (III):

wherein:

-   -   the dotted circle identifies an active moiety;    -   m and n are integers from 1 to 4;    -   B is a substituted or unsubstituted mono or polycyclic aryl or        mono or polycyclic heterocyclyl or heteroaryl with each cyclic        unit containing from 1 to 5 heteroatoms selected from the group        consisting of nitrogen, sulfur, and oxygen;    -   R_(1c) and R_(2c) are independently H, OH, OR_(3c), halogen, CN,        NO₂, COOH, NH₂, NHR_(3c), NR_(3c)R_(4c), C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen; and    -   R_(3c) and R_(4c) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl containing from 1 to 5 heteroatoms selected from the        group consisting of nitrogen, sulfur, and oxygen; and

(iv) a compound comprising the active moiety of formula I, formula II,or formula III. Glutaminase C is then contacted with the compound underconditions effective to reduce the production of glutamate fromglutamine in a cell or a tissue.

The compounds described in the present invention may further comprise anactive moiety (linkable to other moieties), where the active moiety hasthe formula:

Exemplary compounds of the present invention include any of thefollowing:

Another aspect of the present invention relates to a method of treatinga subject with a condition mediated by production of glutamate fromglutamine. The method involves selecting a subject with a conditionmediated by production of glutamate from glutamine by glutaminase C andadministering to said selected subject an inhibitor of glutaminase Cactivity under conditions effective to treat the condition mediated byproduction of glutamate from glutamine.

The inhibitor according to this aspect of the present invention may bean inhibitor of expression-independent glutaminase C activity and/or aninhibitor of glutaminase C activity independent of exogenous phosphateaddition. Alternatively, phosphorylation of glutaminase C can beinhibited.

This treatment can be carried out for the benefit of humans or animals(e.g. rat, mice, pigs, horses, monkeys, cows, sheep, guinea pigs, dogs,and cats).

Suitable examples of such inhibitors include any of the compoundsdescribed above.

The compounds of the present invention can be administered, e.g., byintravenous injection, intramuscular injection, subcutaneous injection,intraperitoneal injection, topical, sublingual, intraarticular (in thejoints), intradermal, buccal, ophthalmic (including intraocular),intranasally (including using a cannula), or by other routes. Thecompounds of the present invention (e.g., formulae I, II, and/or III (aswell as compounds comprising their active moieties)) can be administeredorally, e.g., as a tablet or cachet containing a predetermined amount ofthe active ingredient, gel, pellet, paste, syrup, bolus, electuary,slurry, capsule, powder, granules, as a solution or a suspension in anaqueous liquid or a non-aqueous liquid, as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion, via a micellar formulation(see, e.g. WO 97/11682, which is hereby incorporated by reference in itsentirety) via a liposomal formulation (see, e.g., European Patent No.736299, WO 99/59550, and WO 97/13500, which are hereby incorporated byreference in their entirety), via formulations described in WO03/094886, which is hereby incorporated by reference in its entirety, orin some other form. The compounds of the present invention can also beadministered transdermally (i.e. via reservoir-type or matrix-typepatches, microneedles, thermal poration, hypodermic needles,iontophoresis, electroporation, ultrasound or other forms ofsonophoresis, jet injection, or a combination of any of the precedingmethods (Prausnitz et al., Nature Reviews Drug Discovery 3:115 (2004),which is hereby incorporated by reference in its entirety). Thecompounds can be administered locally, for example, at the site ofinjury to an injured blood vessel. The compounds can be coated on astent. The compounds can be administered using high-velocity transdermalparticle injection techniques using the hydrogel particle formulationdescribed in U.S. Patent Publication No. 20020061336, which is herebyincorporated by reference in its entirety. Additional particleformulations are described in WO 00/45792, WO 00/53160, and WO 02/19989,which are hereby incorporated by reference in their entirety. An exampleof a transdermal formulation containing plaster and the absorptionpromoter dimethylisosorbide can be found in WO 89/04179, which is herebyincorporated by reference in its entirety. WO 96/11705, which is herebyincorporated by reference in its entirety, provides formulationssuitable for transdermal administration.

The condition mediated by production of glutamate from glutamineinclude, without limitation, breast cancer, lung cancer, brain cancer,pancreatic cancer, and colon cancer.

Another aspect of the present invention relates to a pharmaceuticalcomposition comprising a compound selected from the group consisting of:

(i) a compound of formula (II):

wherein:

-   -   n is an integer from 1 to 4;    -   R_(1b) is independently at each occurrence H, OH, OR_(5b),        halogen, CN, NO₂, NH₂, NHR_(5b), NR_(5b)R_(6b), C₁-C₆ alkyl,        C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen;    -   R_(2b) is independently H, halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl,        C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl, or mono        or polycyclic aryl;    -   R_(3b) and R_(4b) are independently H, OR_(5b), SR_(5b),        R_(5b)S(O)—, R_(5b)S(O)₂—, —COOR_(5b), —C(O)NR_(5b)R_(6b), C₁-C₆        alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇        cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or        mono or polycyclic heteroaryl with each cyclic unit containing        from 1 to 5 heteroatoms selected from the group consisting of        nitrogen, sulfur, and oxygen; or    -   R_(3b) and R_(4b) can combine together to form a mono or        polycyclic heterocyclyl or heteroaryl containing from 1-5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each formed heteroaryl or heterocyclyl        optionally substituted with substituents selected from the group        consisting of oxo, thio, amino, C₁-C₆ alkyl, C₂-C₆ alkenyl, and        C₂-C₆ alkynyl; and    -   R_(5b) and R_(6b) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen, each one of R_(5b) or R_(6b) optionally        substituted from 1 to 3 times with substituents selected from        the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆        alkynyl, C₃-C₆ cycloalkyl, and C₄-C₇ cycloalkylalkyl;

(ii) a compound of formula (III):

wherein:

-   -   m and n are integers from 1 to 4;    -   B is a substituted or unsubstituted mono or polycyclic aryl or        mono or polycyclic heterocyclyl or heteroaryl with each cyclic        unit containing from 1 to 5 heteroatoms selected from the group        consisting of nitrogen, sulfur, and oxygen;    -   R_(1c) and R_(2c) are independently H, OH, OR_(3c), halogen, CN,        NO₂, COOH, NH₂, NHR_(3c), NR_(3c)R_(4c), C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl with each cyclic unit containing from 1 to 5        heteroatoms selected from the group consisting of nitrogen,        sulfur, and oxygen; and    -   R_(3c) and R_(4c) are independently H, C₁-C₆ alkyl, C₂-C₆        alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇ cycloalkylalkyl,        aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono or polycyclic        heteroaryl containing from 1 to 5 heteroatoms selected from the        group consisting of nitrogen, sulfur, and oxygen; and

(iii) a compound comprising the active moiety of formula II or formulaIII.

According to this aspect of the present invention, the pharmaceuticalcompositions can comprise a compound of the present invention and apharmaceutically acceptable carrier and, optionally, one or moreadditional active agent(s) as discussed below.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, aformulation intended for the oral administration of humans may vary fromabout 5% to about 95% of the total composition. Dosage unit forms willgenerally contain between from about 1 mg to about 500 mg of activeingredient.

Any pharmaceutically acceptable liquid carrier suitable for preparingsolutions, suspensions, emulsions, syrups and elixirs may be employed inthe composition of the invention. Compounds of the present invention maybe dissolved or suspended in a pharmaceutically acceptable liquidcarrier such as water, an organic solvent, or a pharmaceuticallyacceptable oil or fat, or a mixture thereof. The liquid composition maycontain other suitable pharmaceutical additives such as solubilizers,emulsifiers, buffers, preservatives, sweeteners, flavoring agents,suspending agents, thickening agents, coloring agents, viscosityregulators, stabilizers, osmo-regulators, or the like. Examples ofliquid carriers suitable for oral and parenteral administration includewater (particularly containing additives as above, e.g., cellulosederivatives, preferably sodium carboxymethyl cellulose solution),alcohols (including monohydric alcohols and polyhydric alcohols, e.g.,glycols) or their derivatives, or oils (e.g., fractionated coconut oiland arachis oil). For parenteral administration the carrier may also bean oily ester such as ethyl oleate or isopropyl myristate.

Pharmaceutically acceptable salts include, but are not limited to, aminesalts, such as but not limited to, N,N′-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine, and tris (hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to, lithium, potassium, andsodium; alkali earth metal salts, such as but not limited to, barium,calcium, and magnesium; transition metal salts, such as but not limitedto, zinc; and other metal salts, such as but not limited to, sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited to,hydrochlorides and sulfates; and salts of organic acids, such as but notlimited to, acetates, lactates, malates, tartrates, citrates,ascorbates, succinates, butyrates, valerates and fumarates.Pharmaceutically acceptable esters include, but are not limited to,alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclylesters of acidic groups, including, but not limited to, carboxylicacids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinicacids, and boronic acids. Pharmaceutical acceptable enol ethers include,but are not limited to, derivatives of formula C═C(OR) where R ishydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, orheterocyclyl. Pharmaceutically acceptable enol esters include, but arenot limited to, derivatives of formula C═C(OC(O)R) where R is hydrogen,alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.Pharmaceutical acceptable solvates and hydrates are complexes of acompound with one or more solvent or water molecules, or 1 to about 100,or 1 to about 10, or one to about 2, 3, or 4, solvent or watermolecules.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health, sex, diet time of administration, route ofadministration, rate of excretion, drug combination and the severity ofthe particular disease undergoing therapy.

All methods comprise administering to the subject in need of suchtreatment an effective amount of one or more compounds of the presentinvention.

A subject or patient in whom administration of the therapeutic compoundis an effective therapeutic regimen for a disease or disorder ispreferably a human, but can be any animal, including a laboratory animalin the context of a clinical trial or screening or activity experiment.Thus, as can be readily appreciated by one of ordinary skill in the art,the methods, compounds and compositions of the present invention areparticularly suited to administration to any animal, particularly amammal, and including, but by no means limited to, humans, domesticanimals, such as feline or canine subjects, farm animals, such as butnot limited to bovine, equine, caprine, ovine, and porcine subjects,wild animals (whether in the wild or in a zoological garden), researchanimals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats,etc., avian species, such as chickens, turkeys, songbirds, etc., i.e.,for veterinary medical use.

The compounds of the present invention can be administered alone or asan active ingredient of a formulation. Thus, the present invention alsoincludes pharmaceutical compositions of one or more compounds offormulae I, II, and/or III (as well as compounds comprising their activemoieties) containing, for example, one or more pharmaceuticallyacceptable carriers. The compounds of the present invention can beadministered in a form where the active ingredient is substantiallypure.

Numerous standard references are available that describe procedures forpreparing various formulations suitable for administering the compoundsaccording to the invention. Examples of potential formulations andpreparations are contained, for example, in the Handbook ofPharmaceutical Excipients, American Pharmaceutical Association (currentedition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman andSchwartz, editors) current edition, published by Marcel Dekker, Inc., aswell as Remington's Pharmaceutical Sciences (Arthur Osol, editor),1553-1593 (current edition), which are hereby incorporated by referencein their entirety.

Yet another aspect of the present invention relates to a compound offormula:

Another aspect of the present invention relates to the method ofscreening for compounds capable of reducing the production of glutamatefrom glutamine. The method involves providing a cell or tissue underconditions effective for the cell or tissue to produce glutamate fromglutamine as a result of glutaminase C activity. A plurality ofcandidate compounds is provided to contact the cell or tissue and thecandidate compounds which inhibit glutaminase C activity as a result ofsaid contacting is identified.

In one embodiment of this aspect of the present invention, glutaminase Cactivity refers to phosphorylation of glutaminase C.

EXAMPLES

The Examples set forth below are for illustrative purposes only and arenot intended to limit, in any way, the scope of the present invention.

Example 1—Identification of Glutaminase as the Target of 968

Compound 968(5-[3-bromo-4-(dimethylamino)phenyl]-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[a])was obtained from SPECS (Netherlands; CAS registry #311795-38-7). Inorder to identify the molecular target of 968, its active moiety(3-bromo-4-(dimethylamino)benzyl) (See FIG. 1C) (ChemBridge Corporation,San Diego; CAS registry #56479-63-1) was incorporated into biotinhydrazide by reacting 3-bromo-4-(dimethylamino)benzaldehyde orformaldehyde (as a negative control) at a 5-fold molar excess overnightat 42° C., followed by reduction with cyanoborohydride coupling buffer.The 968-biotin adduct (MW=554 Da) was confirmed by mass spectrometry andincubated with streptavidin-agarose beads equilibrated with cell lysisbuffer (5 μM MgCl₂, 120 μM NaCl, 10 μM HEPES, pH 7.4, 0.5% NP-40, 10mg/ml leupeptin, and 10 mg/ml aprotinin) prior to incubation withlysates from NIH 3T3 cells stably expressing Cdc42(F28L) (5 mlcontaining ˜2 mg/ml total protein) for 2 h at 4° C. The beads werewashed 3× with cold lysis buffer and pelleted by centrifugation and theassociated proteins were resolved by SDS-PAGE. A silver-stained proteinband (M_(r)˜66 kDa) that bound specifically to the 968-biotin beads andnot to the control beads was excised and analyzed by mass spectrometryat the Harvard Microchemistry Facility (Cambridge, Mass.) and identifiedas mouse KGA isoform-2 (accession number NP_001106854), the mouseortholog of the human GAC isoform.

Example 2—Mitochondrial Preparations

Mitochondrial preparations were obtained using the mitochondriaisolation kit from QIAGEN (Cat #37612). A suspension containing 2×10⁷cells was transferred into a 50 ml conical tube and centrifuged at 500×gfor 10 minutes at 4° C. The pellets were resuspended in 2 ml of ice-coldlysis buffer (supplied by QIAGEN) and incubated for 10 minutes at 4° C.using an end-over-end shaker. The lysates were centrifuged at 1000×g for10 minutes at 4° C., and the pellets were resuspended in a buffersupplied by the manufacturer and disrupted completely by using ablunt-ended, 23-gauge needle and a syringe, followed by centrifugationat 6000×g for 20 minutes at 4° C. The pellets were resuspended in 100 mlof 20 μM Hepes, pH 7.4, 150 μM NaCl, 1% NP-40, 20 μMb-glycerolphosphate, 1 μM sodium orthovanadate, and 20 μM sodiumfluoride and assayed for GA activity as previously described (Kenny etal., “Bacterial Expression, Purification and Characterization of RatKidney-Type Mitochondrial Glutaminase,” Protein Expr. Purif. 31:140-148(2003), which is hereby incorporated by reference in its entirety) andfurther outlined below for assaying recombinant enzyme, except that therecombinant protein was replaced by 20 μl of resuspended mitochondriallysate.

Example 3—RNAi

All knock-downs were performed by using Stealth Select RNAi Duplexesfrom Invitrogen that were transiently transfected into cells usingLipofectamine 2000. A non-specific oligonucleotide was used as anegative control. The relative knock-down efficiencies were determinedusing the following antibodies: A polyclonal antibody that recognizesboth isoforms of KGA, an anti-RhoC polyclonal antibody from Santa Cruz,an anti-RhoA monoclonal antibody, and an anti-p65/RelA polyclonalantibody from Cell Signaling.

Example 4—Assays of Recombinant Glutaminase Activity

Glutaminase activity assays were performed on recombinant enzyme aspreviously described (Kenny et al., “Bacterial Expression, Purificationand Characterization of Rat Kidney-Type Mitochondrial Glutaminase,”Protein Expr. Purif. 31:140-148 (2003), which is hereby incorporated byreference in its entirety). A plasmid encoding mouse GAC (residues128-603) was cloned into the pET28a vector and the protein was expressedwith an N-terminal histidine (His)-tag. The tag was cleaved usingthrombin and the protein was further purified by anion-exchange andgel-filtration chromatography. Recombinant GAC (1 μM) was incubated withvarying concentrations of 968 in 57 μM Tris-Acetate (pH 8.6) and 0.225μM EDTA by rotating at 37° C. for 30 minutes, in a final volume of 80μl. Compound 968 was diluted in DMSO such that the volume added wasconstant (5 μl) for all samples, ensuring that the concentration of DMSO(6.3% v/v) was the same in each of the assay incubations. A glutaminesolution was then added to give a final volume of 115 μl and a finalconcentration of 17 μM. The reaction proceeded at 37° C. for 1 h and wasstopped by adding 10 μl of ice-cold 3M HCl. An aliquot of the quenchedreaction mixture (10 μl) was added to an incubation containing 114 μMTris-HCl (pH 9.4), 0.35 μM ADP, 1.7 μM NAD and 6.3 U/ml glutamatedehydrogenase to give a final volume of 228 μl. The reaction mixture wasincubated at room temperature for 45 minutes and the absorbance at 340nm was recorded for each sample against a water blank. The absorbance ofthe sample with just the cocktail mixture was subtracted from eachreading to calculate the activity of the enzyme.

Example 5—Glutaminase C Expression

Serial slides of a breast tissue array were obtained from Biomax U.S.A.and were probed with either an antibody against GAC, or an antibodyagainst actin (control). The expression of GAC was then normalized tothe expression of actin for each sample. An increase in GAC proteinlevels were observed in transformed breast tissues. See FIG. 12.

Example 6—Comparison of Glutaminase C and Kidney-Glutaminase Expression

Total RNA was extracted from normal or cancerous breast tissues, andcomplementary DNA was then synthesized. Quantitative PCR (qPCR) wasperformed in triplicate using primer sets to amplify KGA, GAC or GAPDH,the normalizer/housekeeping gene, on an ABI7500 Fast Real-Time PCRSystem. Relative quantification studies were performed with the ABI7500Fast System Sequence Detection Software. See FIG. 13.

NIH 3T3 cells expressing a constitutively active form of Cdc42, Cdc42(F28L), were transiently transfected with either DNA encoding GAC orKGA. The cells were then allowed to grow under conditions permissive forfocus formation and the number of foci were then counted and scored. SeeFIG. 14.

Example 7—Glutaminase C Phosphorylation

NIH 3T3 cells or NIH 3T3 cells stably expressing the Dbl oncogene, weretransiently transfected with DNA encoding a V5-tagged GAC. The cellswere then harvested, and the ectopically expressed GAC was isolated byimmunoprecipitation via the V5 tag. The V5-GAC obtained from one of theDbl samples was additionally treated with alkaline phosphatase underdephosphorylation conditions. The samples were then subjected to 2-D gelanalysis to separate the V5-GAC by charge and size, and the V5-taggedGAC was visualized by Western blotting using an anti-V5 antibody.Multiple modification states of GAC were detected on GAC isolated fromNIH 3T3 cells expressing Dbl as compared to the GAC isolated fromuntransformed NIH 3T3 cells. The multiple modification states of GACwere reversed when the protein was treated with alkaline phosphatase,suggesting that the modifications are phosphorylations. See FIG. 15.

Example 8—Relationship of Glutaminase C Phosphorylation to BasalGlutaminase Activity

NIH 3T3 cells or NIH 3T3 cells stably expressing the Dbl oncogene, weretransiently transfected with DNA encoding a V5-tagged GAC. The cellswere then harvested and the ectopically expressed GAC was isolated byimmunoprecipitation via the V5 tag. The V5-GAC obtained from one of theDbl samples was additionally treated with alkaline phosphtase underdephosphorylation conditions. See FIG. 16. The samples were then assayedfor glutaminase activity in the absence of phosphate (top panel) and therelative expression levels of V5-GAC was determined by Western blottingusing an anit-V5 antibody (bottom panel). The dephosphorylation of GACisolated from Dbl cells resulted in a 75% reduction of basal glutaminase(phosphate independent) activity.

Example 9—Inhibition of Glutaminase C Phosphorylation by Compound 968

NIH 3T3 cells stably expressing the Dbl oncogene, were transientlytransfected with DNA encoding a V5-tagged GAC, and then one sample wastreated with 968 (10 μM) for 48 hours. The cells were then harvested andthe ectopically expressed GAC was isolated by immunoprecipitation viathe V5 tag. The samples were then subjected to 2-D gel analysis toseparate the V5-GAC by charge and size, and the V5-tagged GAC wasvisualized by Western blotting using an anti-V5 antibody. The treatmentof cells with 968 resulted in the significant reduction of at least onephosphorylation state of GAC. Since 968 inhibits the enzymatic activityof GAC, and the phosphorylation appears to be required for its basalenzyme activity, it appears that 968 might be functioning to inhibitglutaminase C by inhibiting the ability of at least one site onglutaminase C to become phosphorylated. See FIG. 17.

Example 10—Effect of Compound 968 on Oncogenic Growth

In cancer cells, GAC undergoes a phosphorylation event(s) which in notobserved in nontransformed cells (left panel). This phosphorylationleads to a phosphate-independent (basal) activation of GAC, resulting ina rise in glutamate production which feeds the TCA cycle to supply thecancer cell with the energy and metabolic intermediates it needs tosupport tumorogenic growth. It is proposed that 968 may function byblocking a tumor-specific phosphorylation event on GAC which isnecessary for its phosphate-independent activity (right panel). Theinhibition of GAC reduces the influx of glutamate into the TCA cycleand, thus, effectively “starves” the tumor cell of needed energy andmetabolic intermediates. See FIG. 18.

Example 11—Inhibition of GAC (Glutaminase C) Activity by Compound 968and B-968

NIH 3T3 cells stably expressing oncogenic Dbl were transientlytransfected with DNA encoding V5-tagged GAC, and cells were treated witheither 968 or BA-968 (10 mM) as indicated for 48 hours. The cells werethen harvested and the ectopically expressed GAC was isolated byimmunoprecipitation via the V5 tag. See FIG. 19. The samples were thenassayed for glutaminase activity in the absence of phosphate (top panel)and the relative expression levels of V5-GAC was determined by Westernblotting using an anti-V5 antibody (bottom panel).

It is demonstrated here that members of the benzo[a]phenanthridinonefamily block the cellular transformation induced by the Rho family-GEFoncogenic Dbl (Diffuse B-cell lymphoma), as read-out in focus-formingassays or by growth in low serum (FIGS. 1A and 1B). The most effectivemolecule, designated 968, is active at 1-10 μM. The phenyl ring (circledin FIG. 1C) is essential for inhibitory activity, as the moleculedesignated BA-968 is still effective, albeit slightly less potent, inblocking Dbl-induced transformation. Compounds 335 or 384, which lackonly the dimethyl amine or bromine, respectively, show little or noinhibition (FIGS. 1A and 5A). 968 is a more potent inhibitor ofDbl-induced transformation, compared to oncogenic H-Ras, when assayingfocus formation in NIH 3T3 cells (FIGS. 5A and 5B), or growth in lowserum (compare FIGS. 1B and 5C), indicating that the transformingactivities of Rho GTPases are particularly sensitive to this smallmolecule. Treatment with 968 shows no significant effects on the growthor morphology of normal NIH 3T3 cells (FIGS. 1D and 1E).

Mutated Rho GTPases that undergo constitutive GDP-GTP exchange(“fast-cyclers”) mimic many of the actions of Dbl, enabling cells togrow in low serum, form colonies in soft-agar (i.e.anchorage-independent growth), and induce tumor formation when injectedinto immuno-compromised mice (Lin et al., “Specific Contributions of theSmall GTPases Rho, Rac and Cdc42 to Dbl Transformation,” J. Biol. Chem.274:23633-23641 (1999), which is hereby incorporated by reference in itsentirety). Cells transformed by different fast-cycling Rho GTPases wereused to determine whether 968 blocked the signaling activity of aspecific Rho GTPase-target of Dbl, such as RhoC. In fact, 968 inhibitedthe transforming activity of a number of activated Rho GTPase mutants,blocking their ability to stimulate NIH 3T3 cells to form colonies insoft-agar (FIG. 2A) and to grow to high density (FIG. 2B) or under lowserum conditions (FIG. 2C), as well as inhibiting their invasiveactivity (FIG. 2D).

Rho GTPases have been implicated in human breast cancer (Burbelo et al.,“Altered Rho GTPase Signaling Pathways in Breast Cancer Cells,” BreastCancer Res. Treat. 84:43-48 (2004); Valastyan et al., “A PleiotropicallyActing microRNA, miR-31, Inhibits Breast Cancer Metastasis,” Cell137:1032-1046 (2009), which are hereby incorporated by reference intheir entirety). The highly invasive MDA-MB231 cells and SKBR3 cellsrepresent two examples of breast cancer cell lines that exhibithyper-activated RhoA and RhoC compared to normal human mammaryepithelial cells (HMECs), as indicated in pull-down assays using GSTfused to the Rho-binding domain of the effector protein Rhotekin (FIG.6). Compound 968 inhibits the ability of both of these breast cancercells to form colonies in soft agar, as effectively as it blockedDbl-induced colony formation in NIH 3T3 cells (FIG. 2E). Similarly, 968inhibits their growth to high density and in low serum, while havinglittle effect on the growth of HMECs (FIGS. 2F and 2G).

The binding target for compound 968 can be identified by using themolecule active moiety (circled in FIG. 1C) labeled with biotin inaffinity precipitation experiments with streptavidin beads. Thisexperiment leads to the detection of a silver-stained band on SDS-gels,M_(r)˜66 kDa, that can be isolated from Cdc42(F28L)-expressing NIH 3T3cell lysates with the biotin-labeled 968-derivative immobilized tostreptavidin beads, but not with beads alone. Microsequence analysisindicates that this 968-binding partner is the mouse isoform-2 orthologof human glutaminase C (GAC), one of two splice variants of an enzymefound in kidney and other tissues, collectively referred to askidney-type glutaminase (KGA), that catalyzes the hydrolysis ofglutamine to glutamate and ammonium (Curthoys, N. P., “Regulation ofGlutaminase Activity and Glutamine Metabolism,” Annu. Rev. Nutr.15:133-159 (1995), which is hereby incorporated by reference in itsentirety) (FIG. 7). It has been verified that the biotin-labeled activemoiety of 968, when immobilized on streptavidin beads,affinity-precipitates an endogenous protein of M_(r)˜66 kDa that reactedwith an antibody recognizing both isoforms of KGA (FIG. 8), as well asprecipitated ectopically expressed V5-tagged GAC (FIG. 3A, top panel).Compound 968 inhibits the enzymatic activity of purified mouse GACprotein expressed in E. coli, whereas structurally-related compoundslike 335 that are less effective at blocking Dbl-transformation (FIG.1A), also showed little ability to inhibit enzyme activity (FIG. 3A).The inhibition by 968 is neither competitive versus substrate(glutamine) nor inorganic phosphate, an activator of the enzyme (Kennyet al., “Bacterial Expression, Purification and Characterization of RatKidney-Type Mitochondrial Glutaminase,” Protein Expr. Purif. 31:140-148(2003), which is hereby incorporated by reference in its entirety),suggesting that it acts in an allosteric manner (FIGS. 9A-C).

Reducing KGA expression by using siRNAs targeting both of its isoformsinhibits the ability of Cdc42(F28L) to stimulate growth in low serum(FIG. 3B) and colony formation in soft agar (FIG. 10A). Knocking-downKGA expression in control NIH 3T3 cells fails to significantly inhibittheir growth in normal serum (FIG. 10B), consistent with the inabilityof 968 to affect their growth or overall morphology (FIGS. 1D and 1E),whereas it strongly inhibits MDA-MB231 and SKBR3 cells from growing inlow serum (FIG. 3C) and in soft-agar (FIG. 10C). Because 968 blocks thegrowth of transformed/cancer cells by inhibiting glutaminase C activity,it should also eliminate the next step in glutamine metabolism, i.e.,the generation of α-ketoglutarate from the GA-product glutamate.Moreover, this would predict that 968-inhibition can be circumvented byadding a cell-permeable analog of α-ketoglutarate to cells. Indeed, itwas found to be the case in SKBR3 cells when assaying growth in lowserum (FIG. 3D), as well as in Dbl-transformed cells when assayingfocus-formation (FIG. 3E).

Dbl-transformed fibroblasts exhibit much higher basal GA activity (i.e.assayed in the absence of inorganic phosphate) than non-transformed NIH3T3 cells (FIG. 3F). Cdc42(F28L)-expressing cells show basal levels ofGA activity that are lower than those for Dbl-transformed cells, butstill higher than non-transformed cells. The GA activity in control NIH3T3 cells is strongly stimulated by phosphate (˜6-fold), such that itapproaches the maximum phosphate-stimulated activity obtained intransformed cells (FIG. 11A). Treatment of transformed cells with 968inhibits their GA activity, with the basal activity being more sensitivethan the phosphate-stimulated activity (see FIGS. 3F and 11A).

Both MDA-MB231 and SKBR3 cells show significantly higher basal GAactivity, compared to normal HMECs, that is sensitive to 968 (FIG. 3G,top panel; the bottom panel shows that equivalent amounts ofmitochondrial protein were assayed, by using the mitochondrial markerVDAC/Porin (Shimizu et al., “Bcl-2 Family Proteins Regulate the Releaseof Apoptogenic Cytochrome c by the Mitochondrial Channel VDAC,” Nature399:483-487 (1999), which is hereby incorporated by reference in itsentirety)). Inorganic phosphate strongly stimulates the GA activity inHMECs (˜5-fold), and although it is still lower than the maximumactivity measured in MDA-MB231 cells, it is similar to thephosphate-stimulated activity assayed in SKBR3 cells (FIG. 11B).Knock-downs of RhoA and RhoC in SKBR3 cells markedly reduce their basalGA activity, without significantly affecting the direct stimulation ofthe enzyme by phosphate, indicating that the basal enzyme activity inthese breast cancer cells is Rho GTPase-dependent (FIG. 11C).

The expression of GAC is shown to be significantly increased inB-lymphoma and prostate cancer cells and to be necessary for theirproliferation and survival (Gao et al., “c-Myc Suppression of miR-23a/bEnhances Mitochondrial Glutaminase Expression and Glutamine Metabolism,”Nature 458:762-765 (2009), which is hereby incorporated by reference inits entirety). The ectopic expression of GAC alone is insufficient totransform cells (FIG. 4A). However, the transient expression of GAC incells stably expressing Cdc42(F28L) causes a dramatic increase infocus-forming activity, that matches Dbl-transformed cells whichtypically exhibit large numbers of foci and high basal levels of GAactivity, and can be blocked by treatment with 968. When thecatalytically dead GAC(S291A) mutant is co-expressed with Cdc42(F28L),there is no detectable increase in transforming activity compared tothat for Cdc42(F28L) alone (FIG. 4B). Collectively, these findingsdemonstrate the need to reach a threshold level of GA activity toachieve the maximum transforming signal and that increased GACexpression alone is not sufficient for increased basal activity.

MDA-MB231 breast cancer cells show higher KGA expression compared toSKBR3 cells or normal HMECs when using an antibody which recognizes bothenzyme isoforms (FIG. 3C, bottom panel; FIG. 3G, bottom panels), whichlikely accounts for their increased levels of basal (FIG. 3G, top panel)and phosphate-stimulated GA activity (FIG. 11B). However, significantdifferences in KGA expression in Dbl- or Cdc42(F28L)-transformed cellscompared to control cells (FIG. 3F, bottom panel) have not beendetected, consistent with their showing similar levels ofphosphate-stimulated GA activity (FIG. 11A). Likewise, KGA expression inSKBR3 cells is not significantly different from normal HMECs (FIG. 3G,bottom panels), as born out by their similar levels ofphosphate-stimulated GA activity (FIG. 11B). Therefore, the increase inbasal GA activity in SKBR3 cells, as well as in Dbl- and RhoGTPase-transformed fibroblasts, cannot be simply attributed to anup-regulation of enzyme expression.

A clue regarding how GA is activated in these transformed/cancer cellscame from the finding that the treatment of Dbl-transformed cells andSKBR3 breast cancer cells with BAY 11-7082, which blocks NF-kBactivation by inhibiting the upstream kinase IKKb (Pickering et al.,“Pharmacological Inhibitors of NF-κB Accelerate Apoptosis in ChronicLymphocytic Leukemia Cells,” Oncogene 26:1166-1177 (2007), which ishereby incorporated by reference in its entirety), significantly reducestheir basal GA activity (FIGS. 4C and 4D, respectively). NF-κB isactivated by Dbl and various Rho GTPases (Perona et al., “Activation ofthe Nuclear Factor-KB by Rho, CDC42, and Rac-1 Proteins,” Genes Dev.11:463-475 (1997); Joyce et al., “Integration of Rac-DependentRegulation of cyclin D1 Transcription Through a NuclearFactor-KB-Dependent Pathway,” J. Biol. Chem. 274:25245-25249 (1999);Cammarano et al., “Dbl and the Rho GTPases Activate NFκB by IκB kinase(IKK)-Dependent and IKK-Independent Pathways,” J. Biol. Chem.276:25876-25882 (2001), which are hereby incorporated by reference intheir entirety), and is essential for Dbl-transformation (Whitehead etal., “Dependence of Dbl and Dbs Transformation on MEK and NF-kappaBActivation,” Mol. Cell. Biol. 19:7759-7770 (1999), which is herebyincorporated by reference in its entirety) and for the transformedphenotypes of human breast cancer cells (Sovak et al., “Aberrant NuclearFactor-kB/Rel Expression and the Pathogenesis of Breast Cancer,” J.Clin. Invest. 100:2952-2960 (1997), which is hereby incorporated byreference in its entirety). Knocking-down the p65/RelA subunit of NF-kBin Dbl-transformed cells and SKBR3 cells also markedly reduces theirbasal GA activity (FIGS. 4C and 4D), whereas treatment with BAY11-7082or knock-downs of p65/RelA has little or no effect on the directstimulation of the enzyme by phosphate (Figures S7D and 11E).

NF-kB might regulate GA by inducing the expression of a protein thatstimulates its activity through a direct interaction or via apost-translational modification. The latter would be analogous to howthe tyrosine phosphorylation of the M2 isoform of pyruvate kinase hasbeen suggested to influence glycolysis in cancer cells (Christofk etal., “Pyruvate Kinase M2 is a Phosphotyrosine-Binding Protein,” Nature452:181-186 (2008), which is hereby incorporated by reference in itsentirety). Indeed, it has been found that V5-tagged GAC, whenectopically expressed in Dbl-transformed cells followed by itsimmunoprecipitation (IP), exhibits significantly higher activitycompared to V5-GAC IPed from non-transformed NIH 3T3 cells (FIG. 4E).The GA activity IPed from Dbl-transformed cells is inhibited by both 968and BA-968, and is markedly reduced when NF-kB activation is blockedprior to IP, thus consistent with the suggestion that GAC is modified intransformed cells in an NF-kB-dependent manner.

The importance of cellular metabolism in the development of cancer, andin particular, the early observations that tumor cells exhibit enhancedglycolytic activity (i.e. the “Warburg effect”), are receiving renewedattention (DeBerardinis et al., “Beyond Aerobic Glycolysis TransformedCells Can Engage in Glutamine Metabolism that Exceeds the Requirementfor Protein and Nucleotide Synthesis,” Proc. Natl. Acad. Sci. USA104:19345-19350 (2007); Christofk et al., “Pyruvate Kinase M2 is aPhosphotyrosine-Binding Protein,” Nature 452:181-186 (2008), which arehereby incorporated by reference in their entirety). ¹³C-NMR metabolicflux experiments have demonstrated that while proliferating cancer cellsexhibit a pronounced Warburg effect, their TCA cycle remains intact andis driven by glutamine metabolism (DeBerardinis et al., “Beyond AerobicGlycolysis: Transformed Cells Can Engage in Glutamine Metabolism thatExceeds the Requirement for Protein and Nucleotide Synthesis,” Proc.Natl. Acad. Sci. USA 104:19345-19350 (2007), which is herebyincorporated by reference in its entirety). This enables cancer cells tosupply a significant fraction of TCA cycle intermediates as precursorsfor biosynthetic pathways (DeBerardinis et al., “The Biology of Cancer:Metabolic Reprogramming Fuels Cell Growth and Proliferation,” CellMetab. 7:11-19 (2008), which is hereby incorporated by reference in itsentirety), and is consistent with the observations that tumor cellsexhibit increased rates of glutamine metabolism and consume greateramounts of glutamine compared to normal cells (Medina et al., “Relevanceof Glutamine Metabolism to Tumor Cell Growth,” Mol. Cell. Biochem.113:1-15 (1992), which is hereby incorporated by reference in itsentirety). The observation that different transformed cell lines andcancer cells show elevated GA activity in their mitochondria that isdependent on Rho GTPase/NF-kB-signaling provides a mechanism for howthese demands for elevated glutamine metabolism are met. Moreover, theability of the small molecule 968 to inhibit GA activity and influencethe aberrant growth properties of transformed/cancer cells raisesintriguing possibilities for new strategies of therapeutic interventionagainst cancer.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. A method of reducing the production of glutamatefrom glutamine by glutaminase C in a cancerous cell or a canceroustissue, said method comprising: inhibiting glutaminase C activity in thecancerous cell or cancerous tissue under conditions effective to reduceproduction of glutamate from glutamine, wherein said inhibitingcomprises: selecting a cancerous cell or a cancerous tissue, wherein thecancer is characterized by glutaminase C hyperactivity and/orglutaminase C overexpression; providing a compound of formula (I):

wherein: the dotted circle identifies an active moiety; X isindependently —CR_(14a)— or N; R_(1a) is H, OH, OR_(14a), C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, R_(14a)C(O)—, R_(14a)OC(O)—, R_(14a)S(O)—,or R_(14a)S(O)₂—; R₂, R_(3a), R_(4a), R_(5a), and R_(6a) are eachindependently H, halogen, NO₂, OH, OR_(14a), —SR_(14a), NH₂, NHR_(14a),NR_(14a)R_(15a), R_(14a)C(O)—, R_(14a)OC(O)—, R_(14a)C(O)O—, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono orpolycyclic heteroaryl with each cyclic unit containing from 1 to 5heteroatoms selected from the group consisting of nitrogen, sulfur, andoxygen; or R_(2a) and R_(3a), R_(3a) and R_(4a), R_(4a) and R_(5a), orR_(5a) and R_(6a) can combine to form a heterocyclic ring; R_(7a),R_(8a), R_(9a), and R_(10a) are each independently H, OH, NH₂, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl, C₄-C₇cycloalkylalkyl, aryl C₁-C₆ alkyl, mono or polycyclic aryl, or mono orpolycyclic heteroaryl with each cyclic unit containing from 1 to 5heteroatoms selected from the group consisting of nitrogen, sulfur, andoxygen, wherein the aryl, heteroaryl, and aryl C₁-C₆ alkyl areoptionally substituted from 1 to 3 times with substitutents selectedfrom the group consisting of halogen, OH, NH₂, C₁-C₆ alkyl, C₂-C₆alkenyl, C₁-C₆ alkoxy, SH, and C₁-C₆ thioalkyl; and R_(11a), R_(12a),R_(13a), R_(14a), R_(15a), R_(16a), and R_(17a) are each independentlyH, halogen, OH, NO₂, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆cycloalkyl, C₄-C₇ cycloalkylalkyl, aryl C₁-C₆ alkyl, or mono orpolycyclic aryl, wherein each one of R_(11a)-R_(17a) is optionallysubstituted with NH₂, OH, halogen, COOH, NO₂, or CN; and contactingglutaminase C in the cancerous cell or cancerous tissue with saidcompound, wherein the production of glutamate from glutamine in thecancerous cell or cancerous tissue is reduced; and wherein saidinhibiting is carried out under conditions effective to inhibit growthof the cancerous cell or cancerous tissue.
 2. The method of claim 1,wherein the active moiety is:


3. The method of claim 1, wherein said inhibiting is carried out byinhibiting an activating phosphorylation event on glutaminase C.
 4. Themethod of claim 1, wherein said inhibiting is carried out by inhibitingoverexpression-independent glutaminase C activity and/or inhibitingglutaminase C activity independent of exogenous phosphate addition. 5.The method of claim 1, wherein said inhibiting is carried out byinhibiting glutaminase C hyperactivity.
 6. The method of claim 1,wherein the compound is compound 968:


7. The method of claim 6, wherein the cancer is characterized byglutaminase C hyperactivity and glutaminase C overexpression.
 8. Themethod of claim 6, wherein the cancer is characterized by glutaminase Chyperactivity without glutaminase C overexpression.
 9. The method ofclaim 6, wherein the cancer is breast cancer, lung cancer, brain cancer,or colon cancer.
 10. The method of claim 9, wherein the cancer is atriple negative breast cancer.
 11. The method of claim 1, wherein thecancer is characterized by glutaminase C hyperactivity and glutaminase Coverexpression.
 12. The method of claim 1, wherein the cancer ischaracterized by glutaminase C hyperactivity without glutaminase Coverexpression.
 13. The method of claim 1, wherein the cancer is breastcancer, lung cancer, brain cancer, or colon cancer.
 14. The method ofclaim 13, wherein the cancer is a triple negative breast cancer.